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Research Articles: Therapeutics, Targets, and Development

p21 and p27 induction by silibinin is essential for its cell cycle arrest effect in prostate carcinoma cells

¹ Department of Pharmaceutical Sciences, School of Pharmacy; ² Department of Biochemistry and Molecular Genetics; and ³ University of Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado

Requests for reprints: Rajesh Agarwal, Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, 4200 East Ninth Street, Box C238, Denver, CO 80262. Phone: 303-315-1381; Fax: 303-315-6281. E-mail: Rajesh.Agarwal{at}UCHSC.edu

Abstract

Recent studies have shown that silibinin induces p21/Cip1 and p27/Kip1 and G₁ arrest in different prostate cancer cells irrespective of p53 status; however, biological significance and mechanism of such induction have not been studied. Here, using two different prostate cancer cell lines DU145 and 22Rv1, representing androgen-independent and androgen-dependent stages of malignancy, first we investigated the importance of p21 and p27 induction in silibinin-mediated G₁ arrest. Silencing p21 and p27 individually by RNA interference showed marked reversal in G₁ arrest; however, their simultaneous ablation showed additional reversal of G₁ arrest in 22Rv1 but not DU145 cells. These results suggest that whereas relative importance of these molecules might be cell line specific, their induction by silibinin is essential for its G₁ arrest effect. Next, studies were done to examine mechanisms of their induction where cycloheximide-chase experiments showed that silibinin increases p21 and p27 protein half-life. This effect was accompanied by strong reduction in Skp2 level and its binding with p21 and p27 together with strong decrease in phosphorylated Thr¹⁸⁷ p27 without considerable change in proteasomal activity, suggesting a posttranslational mechanism. Skp2 role was further elucidated using Skp2-small interfering RNA–transfected cells, where decreased G₁ arrest and attenuated Cip/Kip induction were observed with silibinin treatment. Further, silibinin caused a marked increase in p21 and p27 mRNA levels together with an increase in their promoter activity, also indicating a transcriptional mechanism. Together, our results for the first time identify a central role of p21 and p27 induction and their regulatory mechanism in silibinin-mediated cell cycle arrest. [Mol Cancer Ther 2007;6(10):2696–707]

Grant support: National Cancer Institute/NIH grant CA102514.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2/15/07; revised 8/ 2/07; accepted 8/30/07.

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